1. Field of the Invention
The present invention relates generally to the fields of biochemistry and molecular biology. More specifically, the present invention relates to the role of p42/44MAPK (also known as extracellular-signal regulated kinase, ERK-1/2) in the induction of low density lipoprotein (LDL) receptor expression.
2. Description of the Related Art
Mitogen-activated protein kinases (MAPK) are cellular signaling pathways that enable cells to transduce extracellular signals into an intracellular response (Robinson and Cobb, 1997; Schaeffer and Weber, 1999). In mammalian cells, three parallel MAPK pathways have been identified. The classical p42/44MAPK pathway is activated in response to signals from cell surface receptors, followed by activation of Raf-1 kinase and MAPK kinase (MEK), which directly activates p42/44MAPK (also known as extracellular-signal regulated kinase, ERK-1/2) through phosphorylation at regulatory threonine and tyrosine residues. In contrast, p38MAPK and p46/54JNK pathways are primarily activated by cellular stress signals such as proinflammatory cytokines, heat shock, or UV light and have therefore also been described as xe2x80x9cstress-activated protein kinasesxe2x80x9d (Whitmarsh and Davis, 1996; Garrington and Johnson, 1999). The signals transmitted through the p42/44MAPK cascade lead to activation of a set of regulatory molecules that play a key role in a variety of cellular responses, including proliferation, differentiation, and cell death. From the published reports, it is evident that the magnitude and duration of p42/44MAPK activation appears to be a key determinant in cell fate signaling (Marshal, 1995; Pumiglia and Decker, 1997; Bornfeldt et al., 1997).
It is now well accepted that relationships exist among the processes of cell growth and those of cholesterol synthesis and metabolism (Habenicht et al., 1984; Fairbank et al., 1984; Casey et al., 1989; Hancock et al., 1989; Gutrierrez et al., 1989; Goldstein and Brown, 1990). Mammalian cells require cholesterol as a structural component of their plasma membrane and other membranes. Cholesterol required for membrane biosynthesis can be derived either from endogenous synthesis within the cell or from an exogenous source. Under normal conditions, many cell types primarily obtain cholesterol from exogenous low density lipoprotein (LDL) via the low density lipoprotein receptor pathway (Brown and Goldstein, 1986).
To investigate the role of the p42/44MAPK signaling cascade in regulating LDL receptor expression, a specific, cell-permeable, noncompetitive inhibitor of MEK-1/2, PD98059 ([2-(2xe2x80x2-amino-3xe2x80x2-methoxyphenyl)-oxanaphthalene-4-one]) (Pang et al., 1995), was utilized. A requirement of the p42/44MAPK cascade during induction of LDL receptor expression in response to a variety of agents, including phorbol-esters, hepatocyte growth factor, interleukin-1xcex2, and anisomycin (Kumar et al., 1997; Kumar et al., 1998; Singh et al., 1999; Dhawan et al., 1999; Mehta and Miller, 1999). Investigations by other laboratories also supported the requirement of the p42/44MAPK signaling cascade in insulin and oncostatin-induced LDL receptor expression (Kotzka et al., 2000; Liu et al., 2000). Taken together, these studies showed that different extracellular signals require signaling through p42/44MAPKto induce LDL receptor expression.
However, given the complexities of cytokine/growth factor signaling, a number of questions were raised by these initial and limited studies, including the following: (i) What is the relationship between p42/44MAPK activation, low density lipoprotein receptor expression and cell growth? (ii) Is activation of p42/44MAPK alone sufficient to induce low density lipoprotein receptor expression, or is participation of other signaling pathways along with p42/44MAPK required for induction of low density lipoprotein receptor expression? (iii) What is the nature of p42/44MAPK-mediated low density lipoprotein receptor induction in terms of sterol sensitivity?
Thus, the prior art is deficient in identifying a direct role of p42/44MAPK in the induction of LDL receptor expression. The present invention fulfills this long-standing need and desire in the art.
The present study reports the results of studies designed to address the relationship between p42/44MAPK activation and IDL receptor expression. A HepG2-derived cell line that stably expresses an inducible form of Raf-1:ER, a fusion protein consisting of an oncogenic form of human Raf-1 kinase (amino acids 305 to 648 that encode all of the kinase domain contained in conserved region 3 but none of conserved regions 1 or 2) and the hormone-binding domain of the human estrogen receptor was generated. It was shown that specific activation of the Raf-1l/MEK/p42/44MAPK cascade by ICI182,780 induces low density lipoprotein receptor expression and modulation of the Raf-1 kinase signal strength is sufficient to determine low density lipoprotein receptor expression levels. Interestingly, Raf-1 kinase activation inhibited DNA synthesis and caused growth arrest, indicating that activation of the Raf-1/MEK/p42/44MAPK cascade uncouples regulation of low density lipoprotein receptor expression from cell growth and these two processes can be regulated independently.
The findings reported here may be of considerable potential significance with regard to the process of low density lipoprotein receptor regulation in vivo. The present findings also underscore the important and central role of the MAPK pathway in regulating LDL receptor expression and may be of considerable potential significance for the development of new signal transduction-based approaches for the treatment of hypercholesterolemia.
In one embodiment of the present invention, there is provided a method of inducing LDL receptor expression through the sole activation of extracellular-signal regulated kinase (p42/44MAPK) by contacting a cell with a compound that activates the extracellular-signal regulated kinase, p42/44MAPK, wherein the activation of said kinase results in the induction of low density lipoprotein receptor expression. Generally, the induction of low density lipoprotein receptor expression is independent of cell growth regulation, whereas the extent of the induction of low density lipoprotein receptor expression is dependent on the extent of activation of p42/44MAPK. Preferably, the cell is the HepG2-xcex94Raf1:ER cell line.
In another embodiment of the present invention, there is provided a method of screening a candidate compound that induces p42/44MAPK-mediated LDL receptor expression by contacting a cell that activates p42/44MAPK in response to extracellular stimulant with a candidate compound, followed by measuring the activation of p42/44MAPK and low density lipoprotein receptor expression. Activation of p42/44MAPK and induction of low density lipoprotein receptor expression in the presence of the compound is indicative of the compound""s ability in inducing p42/44MAPK-mediated low density lipoprotein receptor expression. Preferably, the cell is the HepG2-xcex94Raf-1:ER cell line.
In still another embodiment of the present invention, there is provided a method of determining the level of low density lipoprotein receptor expression in an individual. This method comprises examining the level of p42/44MAPK expression in said individual. In general, high p42/44MAPK expression is indicative of high low density lipoprotein receptor expression, whereas low p42/44MAPK expression is indicative of low LDL receptor expression in said individual.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.